Insulin is an important peptide hormone required to maintain blood glucose homeostasis in mammals, including humans, and other vertebrates. In healthy individuals an increase in blood glucose level stimulates the β-cells of the pancreas to secrete insulin. The insulin polypeptide then binds to specific receptors in muscle, liver, and adipose tissue leading to an increase in glucose uptake by these targeted tissues, an increase in metabolism, and a decrease in hepatic glucose production. The cumulative effects of these responses serve to keep blood glucose concentrations at a constant level.
In individuals suffering from diabetes mellitus, an abnormally low insulin concentration presents itself as chronic hyperglycemia. The clinical manifestations of chronic hyperglycemia are manifold, and include blindness, kidney failure and, if left untreated, will ultimately result in death. Estimates place diabetes mellitus as the third largest cause of death in industrialized countries, after cardiovascular diseases and cancer (Barfoed, H. C., 1987, Chem. Eng. Prog. 83:49-54). In order to allow efficient uptake and metabolism of blood glucose by the cells, diabetic individuals may be treated by the routine administration of insulin. Approximately 0.7% of the world's population suffers from insulin-dependent diabetes (diabetes mellitus Type I) (Winter, J. et al., 2000, J. of Biotechnol. 84:175-185). In addition, it is estimated that the number of individuals diagnosed with diabetes will double to approximately 300 million, in the next 25 years (Kjeldsen, T. et al., 2001, Biotechnol. Gen. Eng. Rev. 18:89-121). Consequently, the ability to cost effectively manufacture human insulin in quantities to satisfy the anticipated growing world demand for insulin is highly desirable.
In vivo the human insulin polypeptide is produced by the pancreatic β-cells as a single 110 amino acid polypeptide chain precursor, preproinsulin, which includes an N-terminally located 24 amino acid pre-sequence that is cleaved immediately upon completion of the chain's biosynthesis (Steiner, D. F. 2000. J. Ped. Endocrinol. Metab. 13:229-239). Proinsulin consists of a B and A chain, linked by a connecting peptide (C-peptide). During packaging of the hormone for secretion the C-peptide is cleaved and removed by prohormone convertases, PC2 and PC1/PC3 (Steiner, D. F. 2000. J. Ped. Endocrinol. Metab. 13:229-239). What remains is mature human insulin, a 51 amino acid protein consisting of two polypeptide chains, A (21 amino acids in length) and B (30 amino acids in length), linked by two inter-chain disulphide bonds. Additionally, the A chain comprises one intra-chain disulphide bond.
Human insulin has been prepared using a variety of different methodologies. Microorganisms such as Escherichia coli (Frank et al., 1981, in Peptides: Proceedings of the 7th American Peptide Chemistry Symposium (Rich & Gross, eds.), Pierce Chemical Co., Rockford. Ill. pp 729-739; Chan et al., 1981, Proc Natl. Acad. Sci. USA 78: 5401-5404), Saccharomyces cerevisiae (Thim et al., 1986, Proc. Natl. Acad. Sci. USA 83: 6766-6770) are routinely employed to recombinantly produce insulin. Wang et al. (Biotechnol. Bioeng., 2001, 73:74-79) have shown that fungi, such as Pichia pastoris, are also suitable for insulin production. Alternative manufacturing options include production in non-human mammalian cell lines (Yanagita, M., et al., 1992, FEBS Lett 311:55-59), isolation from human pancreas, peptide synthesis, or the semisynthetic conversion to human insulin from porcine and bovine insulin. However, all of these methods suffer from lower yields and higher costs than desired.
The use of plants as bioreactors for the large scale production of recombinant proteins is well known, and numerous proteins, including human therapeutic proteins, have been produced. For example, U.S. Pat. Nos. 4,956,282, 5,550,038 and 5,629,175 disclose the production of γ-interferon in plants; U.S. Pat. Nos. 5,650,307, 5,716,802 and 5,763,748 detail the production of human serum albumin in plants and U.S. Pat. Nos. 5,202,422, 5,639,947 and 5,959,177 relate to the production of antibodies in plants. One of the significant advantages offered by plant-based recombinant protein production systems is that by increasing the acreage of plants grown, protein production can be inexpensively scaled up to provide for large quantities of protein. By contrast, fermentation and cell culture systems have large space, equipment and energy requirements, rendering scale-up of production costly. However, despite the fact that the use of plants as bioreactors is amply documented, and despite the above mentioned anticipated prodigious increase in need for large volumes of insulin, the prior art provides only a limited number of methods which demonstrably result in the production of insulin in plants (see: Arakawa et al. Nature Biotech., 1998, 16: 934-938; PCT 01/72959).
Arakawa et al. disclose the production of a fusion protein comprising insulin in the tubers of transgenic potato plants. However insulin represents only up to 0.05% of the total soluble protein content present in the transgenic tubers. At a level of 0.05% of total soluble protein, large amounts of biomass must be subjected to protein extraction rendering the production economics associated with the use of potato tubers unfavorable. Furthermore, Arakawa et al. are not concerned with the isolation of insulin from the potato tuber tissue, but instead suggest an approach prevent the onset of Type I diabetes by inducing immunotolerance which involves oral administration of insulin through the feeding of transgenic potato tubers.
PCT Patent Application WO 01/72959 discloses the production of a fusion protein comprising insulin in chloroplasts of transgenic tobacco. However, while purportedly addressing shortcomings with respect to the accumulation levels of human proteins in plant tissue, the invention to which WO 01/72959 pertains is limited in that production in chloroplasts results in the accumulation of insulin in green tissue, primarily the tobacco leaves. Due to the relatively high water content of green tissue, a large amount of biomass must be processed. Furthermore production of insulin would require immediate extraction from the biomass upon harvesting, as leaf material will rapidly deteriorate when stored.
Thus in view of the shortcomings associated with the methods for the recombinant production of insulin in plants provided by the prior art, it is presently unclear whether and how the synthetic capacity of plants may be harnessed to achieve the commercial production of insulin in plants. There is a need in the art to improve methods for the commercial production of insulin in plants.